Use of dhodh-inhibitors for the treatment of cmarcb1 -mutant associated cancer

Abstract

The invention covers a novel use of DHODH inhibitors for the treatment of cancer in a patient. In particular, there are provided agents and methods for use in the treatment of cancer wherein the level of functional activity of subunits of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex is perturbed,.in the cancer cells is used to determine whether the 5 patient would benefit from treatment with an inhibitor of the DHODH enzyme, and wherein the subunits are the SMARCB1 or SMARCA4 subunits.

Description

[0001] Novel drug combinations for treatment of cancer

[0002] Field of the invention

[0003] The present invention relates to novel and less toxic therapies and methods for the treatment of cancer in a patient. In particular, there are provided agents and methods for use in the treatment of cancer wherein the level of functional activity of SMARCB1 in the cancer cells is used to determine whether the patient would benefit from treatment with an inhibitor of the DHODH enzyme.

[0004] Background

[0005] Malignant rhabdoid tumors (MRTs) are lethal cancers that predominantly strike young children, mostly infants. The vast majority of rhabdoid tumors and MRTs contain bi-allelic inactivating mutations in the SMARCB1 gene (see Roberts & Biegel, 2009, Cancer Biol. Ther. 8(5), 412-416).

[0006] MRTs are highly malignant neoplasms that typically arise in infancy and early childhood. The tumors develop in the brain and spinal cord [referred to as atypical teratoid / rhabdoid tumor (AT / RT)], kidney and / or soft tissues (collectively named extracranial MRT, or ECRT). The histologic appearance of these malignancies can be quite variable. Most tumors contain at least some fields with classic rhabdoid cells, with large nuclei containing a single prominent nucleolus, and cytoplasm with distinct pale eosinophilic inclusions. Tumors demonstrating only classic rhabdoid cells are rare, instead, they often have areas composed of spindled or pleomorphic undifferentiated cells without a rhabdoid phenotype. A classic rhabdoid component may be entirely absent. AT / RTs typically demonstrate a variety of primitive neuroectodermal, epithelial or mesenchymal cells, which underlies the difficulty in distinguishing these tumors from other primitive neuroectodermal tumors or choroid plexus carcinomas. Immunohistochemistry is often used in the differential diagnosis, based on the typical expression of smooth muscle actin, epithelial membrane antigen and vimentin. Lack of expression of the SMARCB1 protein is primarily employed as a specific means of distinguishing rhabdoid tumors from other malignancies with similar histologic features, especially for diagnosis of AT / RT versus primitive neuroectodermal tumors.

[0007] Individuals with germline alterations of SMARCB1 are predisposed to rhabdoid tumors of the brain, kidney and soft tissues such as MRTs, and may present with more than one primary tumor. These children are most often diagnosed within the first year of life and tend to have a worse prognosis. It is not known whether the poor prognosis is related to the presence of a germline mutation in all of their cells, or the fact that they develop multiple and progressive primary tumors that are resistant to therapy. The name SMARCB1 (SWI / SNF related, Matrix associated, Actin dependent Regulator of Chromatin, subfamily B, member 1) is derived from its role as a core member of the SWI / SNF chromatin remodeling complex. SWI / SNF complexes, also known as BRG1 / BRM-associated factor (BAF) complexes, are central regulators of nucleosome remodeling. SWI / SNF complexes are involved in numerous biological processes, including cell cycle regulation and maintenance of genomic stability, and it has been estimated that alterations in SWI / SNF subunits involve over 20% of all cancers. The SMARCB1 protein is also termed SNF5 or INI1 . It is highly conserved, as evidenced by an identical amino acid sequence in mice and humans. However, the function of SMARCB1 is poorly understood. There are no SMARCB1 paralogs and the protein lacks particularly informative protein motifs.

[0008] While there are differing chemotherapeutic regimens for cancers with low or absent activity of SMARCB1 , such as AT / RT, high-dose methotrexate (MTX) is often part of the treatment regimen, and it is also used in other pediatric indications such as pediatric ALL, non-Hodgkin lymphoma, osteosarcoma, and various brain tumors. Despite being part of many pediatric cancer treatment schedules, the use of MTX is associated with severe toxicity and a wide range of side effects. Patients treated with MTX always receive close monitoring of their health and if required also leucovorin rescue therapy. As expected, daily dosing of MTX in ECRT-bearing mice in vivo is associated with toxicity, indicated by significant weight loss over time in some of the mice. Additionally, it has been shown that patients treated with MTX develop resistance for which the biological mechanisms contributing to the generation of this resistance in AT / RT are unknown.

[0009] Therefore, there is a need for the treatment of a cancer characterized by low or absent functional activity of a subunit of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatinremodeling complex, such as MRT. There is further a need for such treatment, without causing severe toxicity and side effects in patients.

[0010] Summary of the invention

[0011] As part of the invention hereunder, we identified targetable metabolic vulnerabilities in MRTs, extra- (ECRTs) and intra-cranial (AT / RTs) MRTs. We also demonstrated that, unexpectedly, targeting MRTs with DHODH inhibitors show in vivo / in vitro efficacy in in hibiting / controlling tumor growth with much less toxicity and side effects than MTX. DHODH inhibitors appear to be safe in the clinical setting although they have not been shown to be effective for the treatment of cancer. For example, BAY-240223423, was discontinued due to lack of benefit for the treatment of myeloid malignancies (available on the internet via www.clinicaltrials.gov / study / NCT03404726). In the invention hereunder, we demonstrate a metabolic dependency to DHODH inhibition in rhabdoid tumors, both in patient-derived organoid (PDO) and in vivo.

[0012] The current invention presents a treatment to decrease the toxicity and side effects caused by the treatment of MRTs, both ECRT and AT / RT, with MTX by using (long-term) DHODH inhibitors (including BAY-2402234). DHODH inhibitors despite having been shown to be safe in various pediatric cancer animal models as well as in a human clinical trial (NCT03404726) for myeloid malignancies, have not yet been shown to be effective for the treatment of cancer. The present invention seeks to provide novel therapies for the treatment of cancers such as rhabdoid tumors, without the severe toxicity and side effects caused by current treatments, as well as methods of selecting an effective treatment regime in cancer patients.

[0013] In some embodiments, the invention provides an inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treating cancer in a patient, wherein the cancer is associated with cells in which the functional activity of SMARCB1 is low or absent.

[0014] In some embodiments, the patient has, or is suspected of having, a cancer selected from the group consisting of malignant rhabdoid tumors, atypical teratoid / rhabdoid tumors, epithelioid sarcomas, synovial sarcomas, undifferentiated sarcomas with or without rhabdoid features, extra skeletal myxoid chondrosarcomas, renal medullary carcinomas, mucinous carcinomas of the pancreas, malignant peripheral nerve sheath tumors, schwannomas, familial and sporadic schwannomatosis, cribriform neuroepithelial tumors, embryonal central nervous system tumors with or without rhabdoid features, choroid plexus carcinomas, teratoma, primitive neuroectodermal tumors, poorly differentiated chordomas, non-Hodgkin lymphoma, and chronic myeloid leukemia. In preferred embodiments, the patient has or is suspected of having malignant rhabdoid tumors (MRT) and / or atypical teratoid / rhabdoid tumors (AT / RT).

[0015] In some embodiments, the patient is assessed prior to commencement of treatment with the inhibitor. In preferred embodiments, the assessment of the patient comprises providing a sample of cells from a patient, and assessing the functional activity of SMARCB1 therein. In some embodiments, the cells are cancer cells. In some embodiments, assessing the functional activity of SMARCB1 comprises measuring the amount of SMARCB1 protein, genomic DNA or cDNA sequence, and / or mRNA encoding the same, in the cells. In preferred embodiments, the assessment of the patient comprises diagnosing the type of cancer from which the patient is suffering.

[0016] In some embodiments, the inhibitor is selected from the list of BAY-2402234, AG636 / AUR- 108, Farudodstat, GTX-196, ASLAN003, JNJ- 74856665, and PTC299. In preferred embodiments, the inhibitor is for administration by a route selected from the group consisting of parenteral, intratumoral, oral, intravenous, transdermal and intramuscular routes. In some embodiments, the inhibitor is for administration at a dose of between 4-500 mg / kg per dose.

[0017] In some embodiments, the invention provides an inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treating cancer in a patient, wherein the cancer is associated with cells in which the functional activity of subunits of the SWItch / Sucrose Non- Fermentable (SWI / SNF) chromatin-remodeling complex is perturbed. In some embodiments, the subunit of the SW / SNF complex represents a SMARC-related subunit. In preferred embodiments, the SMARC-related subunit is SMARCB1 or SMARCA4 and the functional activity of the SMARCB1 or SMARCA4 subunits is low or absent. In some embodiments, the patient has, or is suspected of having, a cancer selected from the group consisting of malignant rhabdoid tumors, atypical teratoid / rhabdoid tumors, epithelioid sarcomas, synovial sarcomas, undifferentiated sarcomas with or without rhabdoid features, ovarian hypercalcaemic small cell carcinomas, non-small cell lung cancer, endometrial undifferentiated carcinoma and sarcoma, gastrointestinal undifferentiated carcinomas, clear cell renal cell carcinoma, prostate cancer, extra skeletal myxoid chondrosarcomas, renal medullary carcinomas, mucinous carcinomas of the pancreas, malignant peripheral nerve sheath tumors, schwannomas, familial and sporadic schwannomatosis, cribriform neuroepithelial tumors, embryonal central nervous system tumors with or without rhabdoid features, choroid plexus carcinomas, teratoma, primitive neuroectodermal tumors, poorly differentiated chordomas, non-Hodgkin lymphoma, and chronic myeloid leukemia. Preferably, the patient has or is suspected of having malignant rhabdoid tumors (MRT) and / or atypical teratoid / rhabdoid tumors (AT / RT).

[0018] In some embodiments, the patient is assessed prior to commencement of treatment with the inhibitor, wherein assessment of the patient comprises providing a sample of cells from a patient and assessing the functional activity of the subunits of the SWI / SNF complex, preferably of the SMARCB1 or SMARCA4 therein. In some embodiments, the cells are cancer cells. In some embodiments, assessing the functional activity of the subunits of the SWI / SNF complex comprises measuring the amount of SMARCB1 or SMARCA4 protein, genomic DNA or cDNA sequence, and / or mRNA encoding the same, in the cells. In preferred embodiments, assessment of the patient comprises diagnosing the type of cancer from which the patient is suffering.

[0019] In some embodiments, the inhibitors are selected from the list of BAY-2402234, AG636 / AUR-108, Farudodstat, GTX 196, ASLAN003, JNJ- 74856665, and PTC299. In preferred embodiments, the inhibitor for administration by a route selected from the group consisting of parenteral, intratumoral, oral, intravenous, transdermal and intramuscular routes. Preferably, the inhibitor is for administration at a dose of between 4-500 mg / kg per dose.

[0020] In some embodiments, the invention provides an inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treating a cancer in a patient, wherein the cancer is characterized by a low or absent functional activity of a subunit of the SWItch / Sucrose Non- Fermentable (SWI / SNF) chromatin-remodeling complex. Preferably, the subunit of the SWI / SNF complex is a SMARC subunit. Preferably, the SMARC subunit is SMARCB1 , SMARCA4, SMARCC1 , SMARCC2, SMARCD1 , or SMARCA2, preferably SMARCB1 or SMARCA4, more preferably SMARCB1. In preferred embodiments, the functional activity is absent.

[0021] In some embodiments, the patient has, or is suspected of having, a cancer selected from the group consisting of malignant rhabdoid tumors (MRT), atypical teratoid / rhabdoid tumors (AT / RT), epithelioid sarcomas, synovial sarcomas, undifferentiated sarcomas with or without rhabdoid features, ovarian hypercalcaemic small cell carcinomas, non-small cell lung cancer, endometrial undifferentiated carcinoma and sarcoma, gastrointestinal undifferentiated carcinomas, clear cell renal cell carcinoma, prostate cancer, extra skeletal myxoid chondrosarcomas, renal medullary carcinomas, mucinous carcinomas of the pancreas, malignant peripheral nerve sheath tumors, schwannomas, familial and sporadic schwannomatosis, cribriform neuroepithelial tumors, embryonal central nervous system tumors with or without rhabdoid features, choroid plexus carcinomas, teratoma, primitive neuroectodermal tumors, poorly differentiated chordomas, nonHodgkin lymphoma, and chronic myeloid leukemia. Preferably, the patient has, or is suspected of having, malignant rhabdoid tumors (MRT) and / or atypical teratoid / rhabdoid tumors (AT / RT).

[0022] In some embodiments, the patient is assessed prior to commencement of treatment with the inhibitor, wherein the assessment of the patient comprises assessing in a sample of cells obtained from the patient the functional activity of a subunit of the SWI / SNF complex, preferably of SMARCB1 or SMARCA4, and wherein low or absent functional activity indicates that treatment with the inhibitor can be commenced. Preferably, the sample of cells are cancer cells. Preferably, assessing the functional activity of the subunit of the SWI / SNF complex comprises at least one of (i) measuring the amount of SMARCB1 or SMARCA4 protein, (ii) measuring the amount of SMARCB1 or SMARCA4 mRNA, and (iii) determining the sequence encoding SMARCB1 or SMARCA4, preferably determining the genomic DNA sequence, cDNA sequence or mRNA sequence encoding SMARCB1 or SMARCA4. Preferably, assessment of the patient comprises diagnosing the type of cancer from which the patient is suffering.

[0023] In some embodiments, the DHODH inhibitor is selected from the group consisting of BAY- 2402234, AG636 / AUR-108, Farudodstat, GTX 196, ASLAN003, JNJ- 74856665, and PTC299, preferably from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, ASLAN003, and PTC299, more preferably PTC299 or BAY-2402234, more preferably BAY-2402234. Preferably, the inhibitor is administered by parenteral, intratumoral, oral, intravenous, transdermal or intramuscular administration, preferably intrathecal or intraventricular administration. Preferably, the DHODH inhibitor is administered at a dose of between 4-500 mg / kg per dose.

[0024] The invention further provides a pharmaceutical formulation comprising the inhibitor for use as described herein, wherein the inhibitor in the formulation has a concentration of between 1 pM and 1 mM.

[0025] The invention further provides a method of treating cancer in a subject, wherein the cancer is characterized by a low or absent functional activity of a subunit of the SWItch / Sucrose Non- Fermentable (SWI / SNF) chromatin-remodeling complex, comprising administering to the subject in need thereof the inhibitor as defined herein, or the pharmaceutical formulation as defined herein.

[0026] Detailed description

[0027] A first aspect of the invention pertains to an inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treating cancer in a patient, wherein the cancer is characterized by low or absent functional activity of one or more subunits of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex. A preferred SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex is a SMARC-subunit, preferably SMARCB1 (SNF5, INI1). Hence in a preferred embodiment, the invention pertains to an inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treatment of a cancer, wherein the cancer is characterized by a low or an absent functional activity of SMARCB1 (SNF5, IN 11 ).

[0028] It is understood herein that the phrase “cancer associated with cells in which the functional activity of subunits of the SWI / SNF chromatin-remodeling complex is perturbed” and the phrase “cancer characterized by a low or absent functional activity of a subunit of the SWI / SNF chromatinremodeling complex” can be used interchangeably herein.

[0029] Cancer

[0030] The term "cancer" is defined as, but is not limited to, a disease associated with expression of a tumor antigen as described herein or condition associated with cells which express a tumor antigen as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen as described herein. The cancer associated with expression of a tumor antigen as described herein can be a hematological cancer. The cancer associated with expression of a tumor antigen as described herein can be is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigen expressing cells express, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen-expressing cells produce the tumor antigen protein (e.g., wild- type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an embodiment, the tumor antigen-expressing cells produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein. In an embodiment, the tumor antigen-expressing cells overexpresses the tumor antigen protein. The terms “a cancer associated with cells” and “a disease associated with cells” can be understood as “a cancer characterized by” and “a disease characterized by”, respectively. The SWI / SNF-complex as described herein can have an effect on one or more tumor antigens.

[0031] The term "cancer" may include a disease associated with expression of a tumor antigen or condition associated with cells which express a tumor antigen including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen. The cancer associated with expression of a tumor antigen can be a hematological cancer. The cancer associated with expression of a tumor antigen can be is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen.

[0032] The term "cancer" includes proliferative diseases. The term cancer may further include a malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia. The cancer can be a hematological cancer. The cancer can be is a solid cancer. The term cancer may further include, but is not limited to, e.g., atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases.

[0033] Preferably, the cancer is a Rhabdoid tumor. The term Rhabdoid tumor (RT) refers to an aggressive (pediatric) soft tissue sarcoma that arises in the kidney, the liver, the peripheral nerves and all miscellaneous soft parts throughout the body. RT involving the central nervous system (CNS) is called atypical teratoid rhabdoid tumor. Atypical teratoid / rhabdoid tumor (AT / RT) of the CNS is an extremely rare and aggressive tumor of - typically - early childhood. The poor outcome with conventional infant brain tumor therapy has resulted in a lack of clear treatment guidelines.

[0034] Central nervous system AT / RTs typically demonstrate a variety of primitive neuroectodermal, epithelial or mesenchymal cells, which underlies the difficulty in distinguishing these tumors from other primitive neuroectodermal tumors or choroid plexus carcinomas. Immunohistochemistry is often used in the differential diagnosis, based on the typical expression of smooth muscle actin, epithelial membrane antigen and vimentin.

[0035] Pediatric kidney tumors mostly encompass Wilms tumors, representing ~80% of cases, but also renal cell carcinomas (RCC), clear cell sarcomas and malignant rhabdoid tumors (extracranial MRT (ecMRT)) can occur. MRT are particularly aggressive malignancies that are typically diagnosed in young children, mostly infants, and can develop throughout the body, including in the brain, where they are called atypical teratoid / rhabdoid tumors (AT / RT). Based on DNA methylation patterns and transcriptome profiles, AT / RT can be further subdivided into three subgroups called MYC, Sonic hedgehog (SHH) and Tyrosinase (TYR). MRT arise as a consequence of derailed differentiation and lineage specification during fetal development, which is caused by the biallelic inactivation of SMARCB1 (~95% of cases) or SMARCA4 (~5% of cases), both of which are SMARC subunits of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex. To date, patient survival is dependent on intensive therapeutic approaches, including chemotherapy, radiotherapy, and surgical intervention. However, in patients that are younger and / or present with metastatic disease, the survival rates remain exceedingly low, highlighting the urgent need for therapeutic innovation.

[0036] In some embodiments, the patient treated with the DHODH inhibitor has, or is suspected of having, a cancer selected from the group consisting of malignant rhabdoid tumors, atypical teratoid / rhabdoid tumors, epithelioid sarcomas, synovial sarcomas, undifferentiated sarcomas with or without rhabdoid features, ovarian hypercalcaemic small cell carcinomas, non-small cell lung cancer, endometrial undifferentiated carcinoma and sarcoma, gastrointestinal undifferentiated carcinomas, clear cell renal cell carcinoma, prostate cancer, extra skeletal myxoid chondrosarcomas, renal medullary carcinomas, mucinous carcinomas of the pancreas, malignant peripheral nerve sheath tumors, schwannomas, familial and sporadic schwannomatosis, cribriform neuroepithelial tumors, embryonal central nervous system tumors with or without rhabdoid features, choroid plexus carcinomas, teratoma, primitive neuroectodermal tumors, poorly differentiated chordomas, non-Hodgkin lymphoma, and chronic myeloid leukemia.

[0037] It is understood herein that the term “tumors” includes one or more tumors. Hence in an embodiment the patient treated with the DHODH inhibitor has, or is suspected of having, a cancer selected from the group consisting of a malignant rhabdoid tumor, an atypical teratoid / rhabdoid tumor, an epithelioid sarcoma, a synovial sarcoma, an undifferentiated sarcoma with or without rhabdoid features, an ovarian hypercalcaemic small cell carcinoma, a non-small cell lung cancer, an endometrial undifferentiated carcinoma, an endometrial undifferentiated sarcoma, a gastrointestinal undifferentiated carcinoma, a clear cell renal cell carcinoma, a prostate cancer, an extra skeletal myxoid chondrosarcoma, a renal medullary carcinoma, a mucinous carcinoma of the pancreas, a malignant peripheral nerve sheath tumor, a schwannoma, familial and sporadic schwannomatosis, a cribriform neuroepithelial tumor, an embryonal central nervous system tumor with rhabdoid features, an embryonal central nervous system tumor without rhabdoid features, a choroid plexus carcinoma, a teratoma, a primitive neuroectodermal tumor, a poorly differentiated chordoma, non-Hodgkin lymphoma, and chronic myeloid leukemia.

[0038] In a preferred embodiment, the patient may have, or may be suspected of having a rhabdoid tumor, preferably an atypical teratoid rhabdoid tumor (AT / RT) and / or a malignant rhabdoid tumour (MRT).

[0039] A subunit of a SWI / SNF complex

[0040] Mammalian SWI / SNF complexes are classified into three subgroups: canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF), also called GLTSCR1 or GLTSCRI L-containing and BRD9-containing (GBAF) complexes. Some subunits and the ATPases are shared between all three subfamilies (e.g., SMARCC1 , SMARCC2, SMARCD1 , SMARCA4, SMARCA2), whereas other components are specific for each subgroup. SMARCB1 participates only into cBAF and PBAF complexes. Preferred SWI / SNF complexes are canonical BAF and polybromo-associated BAF complexes. SWI / SNF complexes are involved in numerous biological processes, including cell cycle regulation and maintenance of genomic stability, and it has been estimated that alterations in SWI / SNF subunits involve over 20% of all cancers.

[0041] SWI / SNF complexes comprise SMARC subunits, such as SMARCB1 , SMARCC1 , SMARCC2, SMARCD1 , SMARCA4, SMARCA2. The terms SMARC subunit and SMARC-related subunit are used interchangeably herein. In an embodiment, the patient is assessed to establish that the cancer is characterized by low or absent functional activity of the SMARC subunit, wherein the subunit is SMARCB1 , SMARCC1 , SMARCC2, SMARCD1 , SMARCA4, or SMARCA2, preferably SMARCB1 or SMARC4, more preferably SMARCB1 .

[0042] In some embodiments, the subunit of the SWI / SNF complex is SMARCB1 , wherein the sequence of SMARCB1 has at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,

[0043] 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 6-9, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0044] In some embodiments, the subunit of the SWI / SNF complex is SMARCB1 , wherein the sequence of SMARCB1 has at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,

[0045] 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NO: 6, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0046] In some embodiments, the subunit of the SWI / SNF complex is SMARCA4, wherein the sequence of SMARCA4 has at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,

[0047] 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 16-20, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0048] In some embodiments, the subunit of the SWI / SNF complex is SMARCA4, wherein the sequence of SMARCA4 has at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,

[0049] 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NO: 16, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0050] SMARCB1 (SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1), a.k.a. INI1 (Integrase Interactor 1), is a subunit ofthe mammalian SWI / SNF (SWItch / Sucrose Non-Fermentable) ATP-dependent chromatin remodeling complexes. SWI / SNF complexes, also known as BRG1 / BRM-associated factor (BAF) complexes, are central regulators of nucleosome remodeling. The protein SMARCB1 is highly conserved, as evidenced by an identical amino acid sequence in mice and humans. However, the function of SMARCB 1 is poorly understood. There are no SMARCB 1 paralogs and the protein lacks particularly informative protein motifs.

[0051] The vast majority of rhabdoid tumors contain bi-allelic inactivating mutations in the SMARCB1 gene. Thus, preferably, the cancer is characterized by bi-allelic inactivating mutations in the SMARCB1 gene. Lack of expression of the SMARCB1 protein is also employed as a specific means of distinguishing rhabdoid tumors from other malignancies with similar histologic features, especially for diagnosis of AT / RT versus primitive neuroectodermal tumors. Individuals with germline alterations of SMARCB1 are predisposed to rhabdoid tumors of the brain, kidney and soft tissues and may present with more than one primary tumor. These children are most often diagnosed within the first year of life and tend to have a worse prognosis. It is not known whether the poor prognosis is related to the presence of a germline mutation in all of their cells, or the fact that they develop multiple and progressive primary tumors that are resistant to therapy. In some embodiments, the cancer is characterized by bi-allelic inactivating mutations in the SMARCB1 gene, wherein the SMARCB1 gene is preferably represented by SEQ ID NO: 1 .

[0052] The “SMARCB1 protein” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 6-9, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0053] The “SMARCB1 protein” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with SEQ ID NO: 6, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0054] The “SMARCB1 mRNA” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 2-5, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0055] The “SMARCB1 mRNA” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with SEQ ID NO: 2, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0056] The “sequence encoding SMARCB1” preferably is a sequence with at least 65, 66, 67, 68,

[0057] 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 1-5, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0058] In some embodiments, the sequence encoding SMARCB1 is the genomic DNA sequence, wherein the genomic DNA sequence is preferably represented by SEQ ID NO: 1. In some embodiments, the sequence encoding SMARCB1 is the mRNA sequence, wherein the mRNA sequence is preferably represented by any one of SEQ ID NOs: 2-5, preferably by SEQ ID NO: 2.

[0059] Similarly, SMARCA4 mutations are the sole gene driving event in almost 100 % of ovarian hypercalcaemic small cell carcinomas, a rare and aggressive form of ovarian cancer. There are also SMARCA4 mutations associated with non-small cell lung cancer , endometrial undifferentiated carcinoma and sarcoma, gastrointestinal undifferentiated carcinomast , clear cell renal cell carcinoma, prostate cancer and other malignancies. In some embodiments, the cancer is characterized by bi-allelic inactivating mutations in the SMARCA4 gene, wherein the SMARCA4 gene is preferably represented by SEQ ID NO: 10.

[0060] Recently, SMARCA4 mutations have been reported in a number of cancers. SMARCA4- deficient thoracic sarcoma (SMARCA4-DTS) occurs predominantly in young men with a history of smoking, and patients usually die within a few months. Other SMARCA4-deficient malignancies include small cell carcinoma, hypercalcaemic type, originally described in the ovary. Cancers consistent with the characteristic morphology and immunophenotype were subsequently reported in the lung, chest, endometrium, sinus tract, gastrointestinal tract and kidney. The prognosis for these diseases is poor and no effective treatment is available. Accordingly, there is a need for therapies for the treatment of cancers such as rhabdoid tumors, as well as method for selecting an effective treatment regime in cancer patients.

[0061] SMARCA4 as used herein is an abbreviation of SWI / SNF related BAF chromatin remodeling complex subunit ATPase 4. SMARCA4 is also known as BRG1 ; CSS4; SNF2; SWI2; MRD16; RTPS2; BAF190; OTSC12; SNF2L4; SNF2LB; hSNF2b; BAF190A; and SNF2-beta. Preferably, SMARCA4 is mammalian SMARCA4, more preferably human SMARCA4.

[0062] The “SMARCA4 protein” preferably comprises a sequence with at least 65, 66, 67, 68, 69,

[0063] 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 16-20, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0064] The “SMARCA4 protein” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with SEQ ID NO: 16, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0065] The “SMARCA4 mRNA” is preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 1 1-15, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0066] The “SMARCA4 mRNA” is preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with SEQ ID NO: 11 , preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%.

[0067] The term “sequence encoding SMARCA4” preferably comprises a sequence with at least 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 95, 96, 97, 98, or 99% sequence similarity with any one of SEQ ID NOs: 10-15, preferably the sequence similarity is at least 75%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, most preferably 100%. In some embodiments, the sequence encoding SMARCA4 is the genomic DNA sequence, wherein the genomic DNA sequence is preferably represented by SEQ ID NO: 10. In some embodiments, the sequence encoding SMARCA4 is the mRNA sequence, wherein the mRNA sequence is preferably represented by any one of SEQ ID NOs: 11-15, preferably by SEQ ID NO: 11 .

[0068] Low or absent functional activity

[0069] Low or absent “functional activity”, is understood herein as a reduced or absent expression of a functional subunit of the SWI / SNF complex, preferably a SMARC subunit, more preferably SMARCB1 or SMARCA4, in a cancer cell. The low or absent “functional activity” is preferably relative to the functional activity of a SWI / SNF complex in a healthy control cell. Hence preferably, the reduced or absent expression of a functional subunit of the SWI / SNF complex, is reduced or absent as compared to the expression of the corresponding SWI / SNF complex in a healthy control cell. In an embodiment, the reduced or absent expression of a SMARC subunit is reduced or absent as compared to the expression of the corresponding SMARC subunit in a healthy control cell. In an embodiment, the reduced or absent expression of SMARCB1 or SMARCA4 is reduced or absent as compared to the expression of respectively SMARCB1 or SMARCA4 in a healthy control cell. Preferably, the healthy control cell is a cell from the same tissue as the cancer cell. Preferably, the healthy control cell is from the same subject as the cancer cell. In some embodiments, the functional activity of a subunit of the SWI / SNF complex in the cancer cell is reduced by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, even more preferably by at least 95%, most preferably by 100%, relative to the functional activity of the corresponding subunit ofthe SWI / SNF complex in the healthy control cell. Preferably, the functional activity of the subunit of the SWI / SNF complex is determined by quantifying the mRNA or protein level of the subunit of the SWI / SNF complex in a cancer cell, and comparing said mRNA or protein level to the mRNA or protein level of the subunit of the corresponding SWI / SNF complex in a healthy control cell.

[0070] In some embodiments, the functional activity of the subunit of the SWI / SNF complex is determined by quantifying the mRNA level of the subunit of the SWI / SNF complex in a cancer cell, and comparing said mRNA level to the mRNA level of the same subunit of the SWI / SNF complex in a healthy control cell. In some embodiments, the mRNA level of the subunit of the SWI / SNF complex in the cancer cell is reduced by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, even more preferably by at least 95%, most preferably by 100%, relative to the mRNA level of the corresponding subunit of the SWI / SNF complex in the healthy control cell. Methods to quantify the mRNA level of a subunit of the SWI / SNF complex are known to the skilled person, and include, but are not limited to, RT-qPCR, next-generation sequencing such as RNA-sequencing, and RNA in- situ hybridization (ISH), of which next-generation sequencing is preferred.

[0071] In some embodiments, the functional activity of the subunit of the SWI / SNF complex is determined by quantifying the protein level of the subunit of the SWI / SNF complex in a cancer cell, and comparing said protein level to the protein level of the corresponding subunit of the SWI / SNF complex in a healthy control cell. In some embodiments, the protein level of the subunit of the SWI / SNF complex in the cancer cell is reduced by at least 50%, more preferably by at least 60%, more preferably by at least 70%, more preferably by at least 80%, more preferably by at least 90%, even more preferably by at least 95%, most preferably by 100%, relative to the protein level of the corresponding subunit of the SWI / SNF complex in the healthy control cell. Methods to quantify the protein level of a subunit ofthe SWI / SNF complex are also known to the skilled person, and include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), western blot, and mass spectrometry (MS), of which MS is preferred. In some embodiments, by low or absent “functional activity”, we mean reduced or absent expression of SMARCB1 or SMARCA4 in tumor cells (for example, relative to internal positive controls in tissue samples, such as normal vessel cells, inflammatory cells, surrounding normal tissue) as assessed at the protein and / or mRNA level, as well as the presence of chromosomal aberrations or DNA mutations (e.g. deletions, miss-sense, nonsense mutations, and the like) or epigenetic alterations (e.g. DNA methylation) that lead to a reduced or lost SMARCBI or SMARCA4 activity. Preferred DNA mutations that lead to a reduced or lost SMARCB1 or SMARCA4 activity are knock-out mutations. Thus, the low or absent functional activity may manifest itself at the level of the genomic DNA, mRNA, protein and / or activity (i.e. function) of SMARCB1 or SMARCA4.

[0072] Assessment of the patient

[0073] It will be appreciated that the assessment step may be performed at any time before or even during treatment of the patient. Preferably, however, the patient is assessed prior to commencement of treatment with the inhibitor.

[0074] In one embodiment, the assessment of the patient comprises providing a sample of cells from the patient and measuring the amount of SMARCB1 or SMARC4 protein, and / or mRNA encoding the same, in the cells. For example, the assessment of the patient may comprise measuring the amount of SMARCB1 or SMARCA4 protein in the cells, eg. by immunohistochemistry, immunofluorescence, Western blot analysis, an immunological assay (e.g., an ELISA or other solid phase-based immunoassay such as SPRIA or amplified ELISA so called IMRAMP), a protein chip assay, surface-enhanced laser desorption / ionization (SELDI), high performance liquid chromatography, mass spectrometry, chemiluminescence, nephelometry / turbometry, lateral flow or pure or polarised fluorescence or electrophoresis.

[0075] It will be appreciated that the sample of cells from the patient may be cancer cells or may be normal (non-cancerous cells). For example, the latter may be useful for detecting the presence of germline mutations associated with or low or absent functional activity of SMARCB1 or SMARCA4.

[0076] Alternatively, or in addition, the assessment of the patient further may comprise measuring the amount of SMARCB1 or SMARCA4 mRNA, e.g. by quantitative PCR, Northern blot analysis, deep sequencing, SAGE, or array technologies.

[0077] Alternatively, or in addition, the assessment of the patient further may comprise determining the level of SMARCB1 or SMARCA4 activity (either directly or indirectly). Such activity may be assayed indirectly, for example by determining the genomic DNA or cDNA sequence, e.g. by fluorescence in situ hybridization, comparative genomic hybridization (CGH), array CGH, other array technologies, or sequencing techniques. The sequence information may then be used to identify chromosomal aberrations or DNA mutations that lead to a reduced or lost SMARCB1 or SMARCA4 activity. Alternatively, or in addition, the assessment of the patient further may comprise determining epigenetic alterations (e.g. DNA methylation, histone modifications), that lead to low or absent SMARCB1 or SMARCA4 gene expression e.g. by DNA methylation analyses, chromatin immunoprecipitation-based techniques, mass spectrometry, chemical reactions (e.g. bisulfite treatment), or methylation profiling used for tumor classification.

[0078] Alternatively, elF2alpha phosphorylation and / or PP1 activity can be used as indirect markers of SMARCB1 or SMARCA4 activity.

[0079] However, it would be apparent to a person skilled in the art that this list of techniques is not complete and these techniques are not the only suitable methods which may be used in the present invention for measuring the functional activity (e.g. expression) of SMARCB1 or SMARCA4.

[0080] Thus, the assessment may comprise performing a biopsy to extract a sample of cancer cells from the patient, which cells can then be tested (either directly or indirectly as a primary cell culture) to determine the functional activity (e.g. expression) of SMARCB1 or SMARCA4 therein. Alternatively, or in addition, normal tissue or cells from the patient may be used to determine the functional activity (e.g. expression) of SMARCB1 or SMARCA4 therein as germline mutations have been found in patients with familial or sporadic tumours.

[0081] However, persons of skill in the art will appreciate that the functional activity (e.g. expression) of SMARCB1 or SMACRA4 may be determined indirectly.

[0082] Thus, the assessment of the patient may comprise diagnosing the type of cancer from which the patient is suffering (using conventional methods well known in the art for cancer diagnosis). This diagnosis can then be used to determine the functional activity (e.g. expression) of SMARCB1 or SMARCA4 in the cancer cells (either through the empirical knowledge of the physician or by consulting a database of gene expression and gene function in known cancer types (such as Gene expression omnibus, ArrayExpress, SAGEmap, RefExA, caArrayData Portal, GeneX, HuGEIndex, TCGA databases, RCGDB, International Cancer Genome Consortium databases, Mitelman database of Chromosome Aberrations and Gene Fusions in Cancer, SKY / M- FISH&CGH database, COSMIC, TmaDB, YMD, dbEST, TMAD, GXA, SMD, Novartis Gene Expression Database, OncoMine and similar databases). Upon determining that the cancer from which the patient is suffering is characterized by (cancer) cells in which the function activity (e.g. expression) of SMARCB1 or SMARCA4 is low or absent, the patient may be administered an inhibitor of the DHODH enzyme as a therapeutic agent to treat the cancer.

[0083] Inhibitor

[0084] By ‘inhibitor of the DHODH enzyme” we mean an agent, such as a small chemical entity, polypeptide or the like, which is capable of inhibiting (at least, in part) a function of the DHODH enzyme (preferably in vivo in humans). Such an inhibitor may act at any point along the DHODH pathway, for example by inhibiting (at least, in part) the enzymes of the de novo nucleotide synthesis pathways. Inhibitors of DHODH enzymes are commonly known to the skilled person. A skilled person is capable of identifying an inhibitor of DHODH enzymes.

[0085] In one embodiment, the inhibitor of the DHODH enzyme is a DHODH inhibitor selected from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, GTX-916, GTX-196, ASLAN003, JNJ- 74856665, and PTC299. Preferably, the DHODH inhibitor is selected from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, GTX-196, ASLAN003, JNJ- 74856665, and PTC299. Preferably, the DHODH inhibitor is selected from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, GTX-196, ASLAN003, and PTC299.

[0086] In one embodiment, the inhibitor of the DHODH enzyme is BAY-2402234. The term BAY- 2402234 is used interchangeably with the term orludodstat. Preferably, the chemical structure of BAY-2402234 is according to CAS number 2225819-06-5.

[0087] In one embodiment, the inhibitor of the DHODH enzyme is AG636 / AUR-108. AUR-108 is formerly known as AG-636, and accordingly, the term AG636 / AUR-108 is understood herein as AG636 and AUR-108. The term AG636 is also known to the skilled person as AG-636. The term AUR-108 is also known to the skilled person as AUR108. Preferably, AG636 / AUR-108 is AG6-636 and / or AUR108. Preferably, the chemical structure ofAG636 / AUR-108 is according to CAS number 1623416-31-8.

[0088] In one embodiment, the inhibitor of the DHODH enzyme is Farudodstat. In one embodiment, the inhibitor of the DHODH enzyme is ASLAN003. The terms Farudodstat and ASLAN003 as used herein can be used interchangeably. Preferably, the chemical structure of Farudodstat is according to CAS number 1035688-66-4.

[0089] In one embodiment, the inhibitor of the DHODH enzyme is GTX-196. GTX-196 is also known as GTX-0196. The structure of this inhibitor is described in US20190152943.

[0090] In one embodiment, the inhibitor of the DHODH enzyme is JNJ- 74856665. Preferably, the chemical structure of JNJ- 74856665 is according to CAS number 2641340-35-2.

[0091] In one embodiment, the inhibitor of the DHODH enzyme is PTC299. PTC299 is also known to the skilled person as Emvododstat. Accordingly, the terms PTC299 and Emvododstat can be used interchangeably. Preferably, the chemical structure of PTC299 is according to CAS number 1256565-36-2.

[0092] In some embodiments, the inhibitor of the DHODH enzyme is selected from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, JNJ- 74856665, and PTC299.

[0093] It will be further appreciated by persons skilled in the art that the inhibitor of the DHODH enzyme may be formulated at various concentrations, depending on a number of factors including the efficacy / toxicity of the inhibitor being used and the indication for which it is being used. Of course, the maximum concentration in any given pharmaceutical formulation will be limited by the maximum solubility of the inhibitor therein. However, the formulations should contain an amount of the inhibitor sufficient to provide an in vivo concentration at or near the target cancer cells which is sufficient to induce their cell death (e.g. via apoptosis).

[0094] In one embodiment, the inhibitor of the DHODH inhibitor is formulated at a concentration of between 1 nM and 1 M. For example, the pharmaceutical formulation may comprise a DHODH inhibitor at a concentration of between 1 uM and 1 mM, for example between 1 uM and 100 pM, between 5 uM and 50 uM, between 10 uM and 50 pM, between 20 uM and 40 uM or about 30 uM.

[0095] Administration and formulation of the inhibitor

[0096] The inhibitors of the DHODH enzyme will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19" edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA; incorporated herein by reference). Suitable routes of administration are discussed below, and include intravenous, oral, pulmonary, intranasal, topical, aural, ocular, bladder and CNS delivery such as, but not limited to, intrathecal and / or intraventricular delivery, for example via an Ommaya reservoir.

[0097] For example, the inhibitor of the DHODH enzyme may be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propyicellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0098] Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and / or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

[0099] The formulations may alternatively be administered parenterally, for example, intravenously, intraarterially, intratumorally, peritumorally, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously (including via an array of fine needles or using needle-free Powderject® technology), or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0100] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or muiti-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

[0101] The inhibitors of the DHODH enzyme may also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetra-fluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA 134A° or 1 ,1 ,1 ,2,3,3, 3-heptaflucropropane (HFA 227EA®), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active DHODH inhibitor, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

[0102] Aerosol or dry powder formulations are preferably arranged so that each metered dose or “puff’ contains at least 1 mg of a compound for delivery to the patient. It will be appreciated that the overall dose with an aerosol will vary from patient to patient and from indication to indication, and may be administered in a single dose or, more usually, in divided doses throughout the day.

[0103] Alternatively, other conventional administration routes known in the art may also be employed; for example the formulation of the invention may be delivered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled- release applications. The formulation may also be administered intra-ocularly, intra- aurally or via intracavernosal injection (see below).

[0104] For application topically, e.g. to the skin, the inhibitor of the DHODH enzyme can be administered in the form of a lotion, solution, cream, gel, ointment or dusting powder (for example, see Remington, supra, pages 1586 to 1597). Thus, the DHODH inhibitors can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, e-lauryl sulphate, an alcohol (e.g. ethanol, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol) and water.

[0105] Formulations suitable for topical administration in the mouth further include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

[0106] The formulation may also be administered by the ocular route, particularly for treating diseases of the eye. For ophthalmic use, the compounds can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

[0107] For veterinary use, a compound is administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

[0108] In one particular embodiment, the formulation is suitable for systemic administration to a patient (for example, via an oral or parenteral administration route).

[0109] The formulation comprising the inhibitor of the DHODH enzyme may be stored in any suitable container or vessel known in the art. It will be appreciated by persons skilled in the art that the container or vessel should preferably be airtight and / or sterilised. Advantageously, the container or vessel is made of a plastics material, such as polyethylene.

[0110] Dose

[0111] The optimal dosage of each combination partner for treatment of a cancer can be determined empirically for each individual using known methods and will depend upon a variety of factors, including, though not limited to, the degree of advancement of the disease; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art. The amount of each combination partner that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.

[0112] Frequency of dosage may vary depending on the compound used and the particular condition to be treated or prevented. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

[0113] An effective dose of the DHODH inhibitor can be based in preclinical studies in mice (PROUS integrity records). The dose escalation study in man will allow to identify the maximum tolerated dose, and will allow to define the recommended clinical dose for pivotal clinical studies.

[0114] An effective dose of the DHODH inhibitor may range from about 4 mg to about 500 mg daily. For example, the DHODH inhibitor may be administered at a dose of between 4 mg / kg and 250 mg / kg, for example between 4 mg / kg and 100 mg / kg, between 4 mg / kg and 50 mg / kg, between 4 mg / kg and 25 mg / kg, between 4 mg / kg and 10 mg / kg or about 8 mg / kg.

[0115] In some embodiments, the inhibitor of DHODH is for administration at a dose sufficient to induce cell death (e.g. apoptosis) of cancer cells in the patient being treated. Thus, the dose of the DHODH inhibitor may be chosen in order to inhibit the growth and / or number of cancer cells in the patient.

[0116] It will be appreciated that the dose of inhibitor of the DHODH enzyme may be changed during the course of treatment of the patient. For example, a higher dose may be used during an initial therapeutic treatment phase of an existing cancer, followed by a lower ‘maintenance’ dose after the initial treatment is complete to prevent recurrence of the cancer.

[0117] In one embodiment, the inhibitor of the DHODH enzyme is for administration at a dose of between 4.0 to 500 mg / kg per dose. For example, the DHODH inhibitor may be administered at a dose of between 4 mg / kg and 250 mg / kg, for example between 4 mg / kg and 100 mg / kg, between 4 mg / kg and 50 mg / kg, between 4 mg / kg and 25 mg / kg, between 4 mg / kg and 10 mg / kg or about 8 mg / kg. The administration may be repeated at regular intervals (for example daily, twice weekly, weekly, bi-weekly, monthly, etc).

[0118] It will be appreciated by persons skilled in the art that the inhibitor of the DHODH enzyme may be for use as a sole treatment for cancer in a patient or as part of a combination treatment (which further treatment may be a pharmaceutical agent, radiotherapy and / or surgery). Thus, the patient may also receive one or more further treatments for cancer, for example pharmaceutical agents (such as chemotherapeutic agents), radiotherapy and / or surgery.

[0119] Definitions

[0120] Various terms relating to the methods, compositions, formulations, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[0121] Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0122] “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article "a" or "an" thus usually means "at least one". Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0123] “About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1 %, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0124] “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0125] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0126] Exemplary": this terms means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations disclosed herein. The term "expression" may refer to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0127] The terms "protein" and "polypeptide" are used interchangeably and may refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds or modified peptide bonds. Polypeptides of less than about 10-20 amino acid residues are commonly referred to as "peptides." The polypeptides of the invention may comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a polypeptide by the cell in which the polypeptide is produced, and will vary with the type of cell. Polypeptides are defined herein, in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.

[0128] The terms “reduced” or “eliminated” or “disruption” or "disrupted" are used interchangeably herein to refer to any genetic modification that decreases or eliminates expression and / or the functional activity of the nucleic acid or an expression product thereof. A preferred expression product is a subunit of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex as defined herein. For example, disruption of a gene includes within its scope any genetic modification that decreases or eliminates expression of the gene and / or the functional activity of a corresponding gene product ( e.g ., mRNA and / or protein). Genetic modifications include complete or partial inactivation, suppression, deletion, interruption, blockage, or down- regulation of a nucleic acid (e.g., a gene). Illustrative genetic modifications include, but are not limited to, gene knockout, inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or use of inhibitory nucleic acids (e.g., inhibitory RNAs such as sense or antisense RNAs, molecules that mediate RNA interference such as siRNA, shRNA, miRNA; etc.), inhibitory polypeptides (e.g., antibodies, polypeptide-binding partners, dominant negative polypeptides, enzymes etc.).

[0129] The term “administering” refers to contacting, applying, injecting, transfusing or providing a composition of the present invention to a subject.

[0130] The term “treating" as used herein may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and / or (4) causing a decrease in the severity of one or more symptoms of the disorder.

[0131] The term “subject” or “patient” as used throughout the specification is to be understood to mean a human or may be a domestic or companion animal. While it is particularly contemplated that the methods of the invention are for treatment of humans, they are also applicable to veterinary treatments, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as primates, felids, canids, bovids, and ungulates. The “subject” may include a person, a patient or individual, and may be of any age or gender.

[0132] Examples

[0133] Example 1 - Introduction, Materials and Methods

[0134] 1. 1 Introduction

[0135] Brain cancer is one of the leading causes of cancer-associated deaths among children [1], Current treatment modalities (eg, surgical resection, intensive chemotherapy, and radiation therapy) have shown limited effectiveness. Moreover, current treatment options often cause nonspecific cytotoxic effects that negatively affect the development of the young patient’s brain, resulting in long-term neurological deficits. In particular, atypical teratoid rhabdoid tumor (AT / RT) is a rare but highly malignant form of brain cancer that predominantly affects children under three years [2,3], The median age at diagnosis is approximately 17 months, and patients typically survive less than one year after diagnosis [2,4], Currently, there is no definitive standard of care for AT / RT, and traditional treatments are not effective enough to stop aggressive progression of the disease. Given the dismal prognosis associated with AT / RT, better treatment approaches are urgently needed to improve the treatment outcomes in these very young patients.

[0136] Despite being part of many pediatric cancer treatment schedules, the use of MTX is associated with severe toxicity and a wide range of side effects. Patients treated with MTX always receive close monitoring of their health and if required also leucovorin rescue therapy. As expected, daily dosing of MTX in ECRT-bearing mice in vivo is associated with toxicity, indicated by significant weight loss over time in some of the mice. Additionally, it has been shown that patients treated with MTX develop resistance for which the biological mechanisms contributing to the generation of this resistance in AT / RT are unknown. Therefore, there is a great medical need to find a treatment of rhabdoid tumors, including MRTs and AT / RTs, without causing this severe toxicity and wide range of side effects in patients.

[0137] As part of the invention herein, we identified targetable metabolic vulnerabilities in MRTs, extra- (ECRTs) and intra-cranial (AT / RTs) malignant rhabdoid tumors. We also demonstrated that, targeting rhabdoid tumors with DHODH inhibitors show in vivo and in vitro efficacy in inhibiting tumor growth with much less toxicity and side effects than MTX.

[0138] The DHODH gene, located in the open reading frame (ORF) of human chromosome 16q22 with full length of 1 191 bp, encodes DHODH protein with 397 amino acid sequences [5], According to sequence similarity and subcellular location, DHODH is divided into Class 1 and Class 2 DHODHs. Soluble class 1 DHODHs are further classified into class 1A, class 1 B, and class 1 S, which are all located in cytoplasm. Class 1A are homodimeric proteins and found in Gram-positive bacteria. Class 1 B DHODHs is a dimer of heterodimers and usually found in prevalent in Grampositive bacteria, consisting of two distinct proteins. The S DHODH is a newly found type which is incapable of utilizing any of the natural electron acceptors. It uses serine as catalytic base, which is special for a cytosolic DHODH [6, 7], Class 2 DHODHs are monomeric proteins that attach to the mitochondrial inner membrane in eukaryotes and some prokaryotes [7-101. Class 1A and class 1 B share approximately 30% sequence identity, whereas soluble class 1 and membrane-bound class 2 DHODHs share approximately 20% of sequence identity [7],

[0139] Pyrimidines are necessary for the biosynthesis of DNA, RNA, glycoproteins and phospholipids [111. Pyrimidine nucleotides are synthesized through two pathways: the de novo synthesis pathway and the salvage pathway [121. Pyrimidines are synthesized de novo from simple precursors with six steps. The enzymes that catalyze uridine monophosphate (UMP) synthesis include carbamoylphosphate synthetase II (CPSII), aspartate transcarbamoylase (ATCase), dihydroorotase (DHOase), DHODH, and uridine monophosphate synthase (UMPS) [131. Firstly, glutamine (Gin), ATP and HCO3- collectively form carbamoyl phosphate, which is catalyzed by CPSII, a vital enzyme located in the cytosol. ATCase contributes to the formation of the carbamoylaspartate following, the pyrimidine ring further is cyclized by DHOase to produce dihydroorotate. Secondly, the flavoenzyme DHODH converts dihydroorotate to orotate which takes place in mitochondria. Orotate further converts to UMP by the UMP synthase UMPS. UMP, a precursor of other pyrimidines and various biological processes, then converts to uridine triphosphate (UTP) by phosphorylation. UTP is subsequently converted to cytidine triphosphate (CTP) with the donor glutamine by CTP synthetase. Meanwhile, the uridine diphosphate (UDP) transforms to deoxyuridine diphosphate (dUDP) by ribonucleotide reductase, with its ribose moiety reducing to deoxyribose. Deoxyuridine monophosphate (dUMP) methylation generates deoxythymine nucleotides (dTMP or TMP) for DNA synthesis [14, 15-171. In this process, DHODH catalyzes the fourth step in the de novo biosynthesis of pyrimidine by converting dihydroorotate into orotate in a redox reaction in the mitochondria with ubiquinone (CoQ) converting to ubiquinol (CoQH2), which is a substrate of respiratory complex III [14, 7, 181.

[0140] As DHODH plays a crucial part in pyrimidine synthesis, it exerts varying effects on different cell types and developmental stages owing to the relative contribution of de novo pyrimidine synthesis to maintaining proliferation. The de novo pyrimidine synthesis has gained a prominent position to satisfy increasing demand for nucleic acid precursors in rapidly proliferative cells such as activated T cells due to the rapidly increased demand for DNA replication and nucleic acid biosynthesis [11 , 15, 19, 20], While in resting or fully differentiated cells, they mainly obtain pyrimidines through the salvage pathway for proliferation [11 , 21-241. Thus, the effect of DHODH is more predominate in rapidly proliferating cells like cancer cells, which may be highly sensitive to inhibition of nucleotide synthesis [25-271.

[0141] Consistent with above observations, DHODH blockade by inhibitors or RNA interference exhibited anti-proliferation effect by pyrimidine depletion. Studies manifest that DHODH inhibitors have more potential to treat malignancies which are more dependent on de novo pyrimidine synthesis and have lower pyrimidine salvage activity. For instance, in PTEN-mutant cells which depend upon glutamine flux through the de novo pyrimidine synthesis pathway

[0028] , inhibition of DHODH causes stalled forks due to inadequate nucleotide pools required to support replication. Sustained treatment with DHODH inhibitor further leads to Rad3-related kinase (ATR) activation, leading to a buildup of DNA damage and cell death

[0028] , Cancer cells are hypersensitive to DHODH inhibitors under the tumor hypoxia and nutrient-deprived microenvironment [19, 29], Further, depletion of the pyrimidine nucleotide pool, especially UTP, resulting from DHODH inhibition, could impair biogenesis of ribosomes, which activates the tumor suppressor p53 pathway leading to cell cycle arrest [30, 311. Moreover, DHODH is regulated by several critical transcription factors [161. The predicted and known regulators of DHODH include E1A-binding protein p300, POU domain, class 3, transcription factor 2 (POU3F2), GATA-binding factor 2 (GATA-2), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NF-KB1), and proto-oncogene MYC

[0016] . It might be significant to inhibit DHODH through interfere with these regulatory factors, which can further contribute to pyrimidine depletion and induce death of tumor cells.

[0142] Apart from the direct blockade of pyrimidine synthesis, DHODH also exerts additional effects including O-linked / V-acetylglucosaminylation (O-GIcNAc)

[0032] , senescence

[0033] , AND mRNA translation [341 indirectly by pyrimidine depletion. UMP, the downstream product of DHODH, is a precursor for components required for the assembly of various cellular macromolecules, including phospholipids, glycogen, hyaluronic acid, and proteoglycans, as well as for certain post- translational protein modifications [23, 35], which may account for the above extensive influences of DHODH. For example, O-GIcNAc, a post-translational modification of proteins, can add N- acetylglucosamine (GIcNAc) on UDP

[0036] , UMP reduction by DHODH suppression may result in integral decreases in protein / V-acetyl glycosylation in acute myeloid leukemia (AML)

[0037] , and reduction of UMP-GIcNAc promotes myeloid differentiation

[0037] , Given that O-GIcNAc plays a crucial role in myeloid differentiation [38-401, it may explain the mechanism that DHODH inhibition promotes the myeloid differentiation in AML to some extent. Collectively, DHODH inhibition decreases the de novo pyrimidine synthesis and affects many biological processes. Despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. In the invention herein, we demonstrate that even though, based on the prior art, the use of DHODH inhibitors would not be an obvious choice for the treatment of cancer, our results in vitro in patient-derived organoid (PDO) and in vivo demonstrated a metabolic dependency to DHODH inhibition in rhabdoid tumors, which was not expected, targetable metabolic vulnerabilities in extra- (ECRTs) and intra-cranial (AT / RTs) malignant rhabdoid tumors. Targeting of de novo pyrimidine synthesis via DHODH inhibitors show in vivo / in vitro efficacy in inhibiting / controlling tumor growth. In xenografts methotrexate (MTX) has been tested. MTX- and BAY-2402234 induced cytotoxicity seems to be tumor-specific in vitro.

[0143] Although genomic instability is a common feature of most malignant cells

[0041] , AT / RT genomes are highly stable. Most AT / RTs contain less than ten coding mutations, and large chromosome gain or loss is uncommon [42-44], The only recurring molecular abnormality that characterizes AT / RT is the inactivation of the SMARCB1 gene, which is observed in nearly 98% of AT / RT patients

[0144]

[0045] , SMARCB1 encodes a core subunit of the SWI / SNF chromatin remodeling complex that contributes to developmental processes. Although up to 20% of human cancers contain a loss-of- function mutation in the SWI / SNF complex, its role in tumor suppression remains understudied

[0145]

[0046] , It was only recent that several studies have revealed that SMARCB1 is a tumor suppressor with its loss required for rhabdoid tumorigenesis [3,46], The precise mechanism through which SMARCB1 can act as a tumor suppressor remains to be fully understood [43,46-50], In rhabdoid tumors, inactivation of SMARCB1 can drive proliferation by altering the expression of multiple pro- oncogenic pathways [51-57], Importantly, loss of SMARCB1 also reduces the efficacy of conventional anti-cancer treatments, such as radiation and chemotherapy [58,59], Thus, restoration of SMARCB1 is an attractive approach for treating patients with AT / RT with the potential to render their AT / RT more sensitive to conventional therapeutic modalities.

[0146] Additionally, SMARCA4-deficient tumors, which represent 5% of MRTs have also shown to be susceptible to DHODH inhibition. Other SMARCA4-deficient malignancies include small cell carcinoma, hypercalcaemic type, originally described in the ovary. Cancers consistent with the characteristic morphology and immunophenotype were subsequently reported in the lung, chest, endometrium, sinus tract, gastrointestinal tract and kidney. The prognosis for these diseases is poor and no effective treatment is available.

[0147] Combining emerging knowledge regarding the role of SMARCB1 , or perturbations in the SWI / SNF complex, in a broad range (rhabdoid) tumors, using the inventors’ expertise and particular models of MRT and AT / RT as well as their knowledge in the role of DHODH inhibition in MRT and AT / RT tumors, the present invention relates to a novel use of DHODH inhibitors for the treatment of cancer in a patient. In particular, there are provided agents and methods for use in the treatment of cancer wherein the level of functional activity of the SWI / SNF complex in the cancer cells is used to determine whether the patient would benefit from treatment with an inhibitor of the DHODH enzyme. Further, there are provided agents and methods for use in the treatment of cancer wherein the level of functional activity of SMARCB1 in the cancer cells is used to determine whether the patient would benefit from treatment with an inhibitor of the DHODH enzyme.

[0148] Information retrieved from Ref. 60 and 61 of item 1 .2.

[0149] 1.2 Experimental model and study participant details Animals:

[0150] 8-week-old NOD-Scid IL2Rgnull mice (male and female) were used as acceptors for subcutaneous injections of MRT organoids. Mice were randomized into groups without blinding. Handling injections, tumor measurements and culling of the mice was performed by the animal technicians of Intervention Unit team at the Mouse Cancer Clinic of the Netherlands Cancer Institute. Daily welfare check was performed by the animal caretakers and technicians of the Mouse Cancer Clinic. Mice were kept under standard temperature and humidity conditions in individually ventilated cages (Innovive), with food and water provided ad libitum.

[0151] Human tissue:

[0152] All organoid models used in this study were previously established by Calandrini et al. (2020), Meister et al. (2022), and Paassen et al. (2023). An overview of the clinical characteristics of the patients from which the models were derived can be found in the Table 1 below.

[0153] Table 1. Clinical characteristics of the tumor samples included in the drug screens and / or metabolomics experiments. Additional clinical information can be retrieved from Calandrini et al. (2020), Meister et al. (2022), and Paassen et al. (2023). *Model derived from the same patient material. NA: information not available. M=male, F=female, y=years, m=months, WT=wild-type, MRT=malignant rhabdoid tumor, AT / RT= atypical teratoid / rhabdoid tumor, RCC= renal cell carcinoma, MYC=subgroup of AT / RT, SHH= sonic hedgehog, a subgroup of AT / RT, TYR=tyrosinase, a subgroup of AT / RT, FN-RMS=fusion-negative rhabdomyosarcoma, FP- RMS=fusion-positive rhabdomyosarcoma, PDOX=patient-derived orthotopic xenograft, Mixed histopathology = blastema, epithelium, and / or stroma

[0154] Patient-derived organoids: patient-derived kidney organoid cultures have been established with protocols previously described by Calandrini et al., 2020. Normal kidney and kidney tumor organoids were cultured in reduced growth factor BME (Cultrex, 3533-010-02) topped with kidney organoid medium (KOM). KOM consists of AdDF+++ (Advanced DMEM / F12 containing 1x GlutaMAX, 10mM HEPES, and antibiotics; Gibco), supplemented with 1.5% B27 supplement (Gibco), 10% R-spondin-conditioned medium, EGF (50 ng / mL, Peprotech), FGF-10 (100 ng / mL, Peprotech), N-acetylcysteine (1.25 mM, Sigma), Rho-kinase inhibitor Y-27632 (10 pM, AbMole), and A83-01 (5 pM, Tocris Bioscience). KOM was changed every 3-4 days, and organoids were passaged every 1-3 weeks. Organoids were either passaged by mechanical dissociation (MRT organoids), or with TrypLE Express (Invitrogen, 1260510) with 10 pM Rho-kinase inhibitor Y-27632 (normal kidney organoids, Wilms tumor organoids, RCC organoids). After adding 5-10 mL AdDF+++ and centrifugation at 300 ref, cells were reseeded in BME and topped with KOM. Atypical teratoid / rhabdoid tumoroid and rhabdomyosarcoma tumoroid cultures were maintained as described by Paassen et al. 2023 and Meister et al. 2022.

[0155] 1.3 Methods

[0156] Bulk RNA sequencing:

[0157] Bulk RNA sequencing on organoids and matching patient tissue was performed as described by Calandrini et al., 2020. For bulk RNA sequencing on normal kidney model 103H and MRT model 103T upon 24-h treatment with DMSO, Methotrexate (MTX) or BAY-2402234 (BAY), 2.500 single cells / pL were seeded in a 6 well suspension plate (Greiner CELLSTAR, cat-no. 657-185) using 300 pL BME droplets per well (75% BME; 25% cell suspension) topped up with 2.5 mL of kidney organoid medium (KOM). Three (103T) to six days (103H) after seeding, medium was removed and exchanged for medium containing DMSO (vehicle), 400 nM MTX (Merck, M1000000), or 50 nM BAY (DC Chemicals, DC23745). After 24 h, RNA was extracted from the organoids using Trizol reagent (Invitrogen), and quality was checked with Bioanalyzer2100 RNA Nano 6000 chips (Agilent, Cat. 5067-1511).

[0158] The NEBNext Ultra RNA Library Prep Kit (New England Biolabs) was used to prepare sequencing libraries. Paired-end sequencing was performed on the Illumina HiSeq or Illumina NovaSeq X Plus by Novogene (Germany).

[0159] Bulk mRNA sequencing kidney data originating from Wilms and Rhabdoid tumors were downloaded from the TARGET database (accessible via the internet via ocg.cancer.gov / programs / target / data-matrix). Paired-end files from each sample were downloaded with SRA-toolkit with parameters that kept only biological reads that passed quality filters and with no tags. After quality control with fastqc, raw files were aligned against human genome hg38 using STAR (v.2.7.2) and read counts quantified with featurecounts (v. 1.6.7) using genome annotation from Gencode v. 37.

[0160] Raw count files from each sample were then merged into a single matrix and processed in R 4.2.1 using the R package DESEq2. Prior to any downstream analysis the count matrix was filtered to keep only genes with at least 5 read counts across all samples, normalized, and transformed into Iog2 scale.

[0161] For both datasets, Gene Ontology analysis was performed using the R package clusterProfiler using a 0.05 q-value cutoff; heatmaps were generated with pheatmap.

[0162] Liquid chromatography-Mass spectrometry (LC-MS)-based metabolomics:

[0163] For the kidney organoids, normal kidney (lines 37H, 57H, 71 H, 117H) and kidney tumor organoids (Wilms lines: 88T, 51 T; MRT lines: 60T, 78T, 103T) were made single cell. For each condition, 0.5x106 cells were plated in triplicate in 75% BME droplets in KOM. When cells had reached confluency, 6 or 24-h labeling with [U-13C6]-glucose (Cambridge Isotopes, CLM-1396-PK) was started. For the LC-MS-based metabolomics experiments with MTX and BAY-2402234 in MRT organoids, 0.2-0.25x106 single cells were immediately after plating treated with either DMSO, 400 nM MTX (Merck, M1000000) or 5 nM BAY-2402234 (DC Chemicals, DC23745) for a duration of 120 h. 96 h after adding the drugs, 24-h labeling with [U-13C6]-glucose was started. For labeling, adDF+++ culture medium was replaced for glucose-free SILAC Advanced DMEM / F-12 (Gibco, A2494301) supplemented with 17 mM [U-13C6]-glucose (Cambridge Isotopes, CLM-1396-PK), 2 mM L-Glutamine (Gibco, 25030081), 10 mM HEPES (Gibco, 15630080), 0.7 mM L-Arginine (Sigma-Aldrich, A5131), 0.5 mM L-Lysine (Sigma-Aldrich, L5626), and antibiotics. Since BME compositions could change after extended culturing, BME containing no cells was plated in triplicate for each tumor type to correct for possible background effects. 6 or 24 h after labeling, the medium was removed and all wells were washed with 1 mL cold PBS, without disrupting the BME droplets. PBS was removed and 500 pL ice-cold MS lysis solvent (LS) composed of methanol / acetonitrile / Milli-Q (2:2:1) was added to each well. Plates were put on a plate Rocker at 4°C for 10min to induce lysis. After 10min, LS was removed from the wells and collected in 1 .5 mL Eppendorf tubes that were then put in a Thermoshaker at 4°C for 15 min. Samples were cleared by centrifugation at 12.000 ref for 15 min. Samples were frozen at -80°C until further use.

[0164] For the AT / RT tumoroids, AT / RT tumoroids and MRT organoids were made single cell by mechanical dissociation, plating 0.5x106 single cells in triplicate per condition in tumor stem medium (TSM, formulation described by Paassen et al. 2023). The next day, 6- or 24-h labeling with [U-13C6]-glucose was started. For labeling, TSM culture medium was replaced for a 50:50 mix of DMEM / F12 without glucose, glutamine and HEPES (Biowest, L0091) and Neurobasal A without glucose and sodium pyruvate (Gibco, A2477501), supplemented with 21.25 mM [U-13C6]-glucose (Cambridge Isotopes, CLM-1396-PK), 2 mM L-Glutamine (Gibco, 25030081), 10 mM HEPES (Gibco, 15630080), 1x MEM non-essential amino acid solution (Gibco, 11140050), 1.5 mM sodium pyruvate (Gibco, 11360070), and the standard growth factors.41 6 or24 h after labeling, the medium was removed, cells were washed with cold PBS and lysed in 75 pL ice-cold LS for 10 min in a 4°C Thermoshaker. After 10 min, samples were cleared by centrifugation at 12.000 ref for 15 min and frozen at -80°C until further use.

[0165] LC-MS analyses of metabolites were performed on a Q Exactive HF mass spectrometer (Thermo Scientific) coupled to a Vanquish autosampler and pump (Thermo Scientific) or Exactive mass spectrometer (Thermo Scientific) coupled to a Dionex Ultimate 3000 autosampler and pump (Thermo Scientific). Sample injection volumes were always 5 pL. The MS operated in polarityswitching mode with spray voltages of -3.5 kV and 4.5 kV. Sheath gas, auxiliary gas, and sweep gas flow rates were 35, 10, and 1 units, respectively. Separation of metabolites was done using a Sequant ZIC-pHILIC column (2.1 x 150 mm, 5 pm; Merck) coupled to a ZIC-pHILIC guard column (2.1 x 20 mm, 5 pm; Merck), using elution buffers acetonitrile for A, and 20mM (NH4)2CO3, 0.1 % NH4OH in LC / MS grade water (Biosolve) for B. Column temperature was 30°C. Flow rates were set at 100 pL / min for the Q Exactive and 150 pL / min for the Exactive and a gradient ran from 80%A to 20%A. Data was analyzed using LCquan or TraceFinder software (Thermo Scientific). Identification and quantification of metabolites was based on exact mass within 5 ppm and validated further by concordance with m / z, retention times and peak shape of reference standards of metabolites of interest that were included in the same run. Peak intensities were normalized based on total ion count and distributions of isotopes were corrected for natural abundance of 13C. The fraction of 13C-labelled metabolites was calculated by dividing the amount of 13C-labeled metabolites ([M+>01]) by the total metabolite pool (sum of labeled [M+>01 ] and unlabeled [M+00] isotopologues) multiplied by 100. The Iog2 fold-change (log2FC) in 13C-labelled fractions was calculated for MTX- and BAY-treated tumoroids relative to DMSO controls. For visualization, the log2FC values were constrained by binning, using a lower threshold of -5 and an upper threshold of 5.

[0166] Drug screens:

[0167] Drug screens on rhabdomyosarcoma (RMS) organoids and atypical teratoid / rhabdoid tumoroids were performed as described by Paassen et al. 2023. For the kidney organoids, 500 cells per well were plated in a 5% BME slurry with KOM in black 384 wells plates (Corning), using a volume of 40 pL per well. Plating of the cells was done with a Multi-drop Combi Reagent 8 Dispenser (Thermo Scientific).

[0168] Methotrexate (Merck, M1000000) and BAY-2402234 (DC Chemicals, DC23745) were added with a Tecan D300e Digital Dispenser. Final concentrations used for drug screenings with Methotrexate were: 0.1 nM, 1 nM, 10 nM, 100 nM, 1 pM and 10 pM, and final concentrations for BAY-2402234 were: 0.01 nM, 0.05 nM, 1 nM, 10 nM, 100 nM and 1 pM. Drug concentrations were normalized by the Tecan D300e dispenser for DMSO content. Cells treated with DMSO served as negative controls. For each organoid model, three independent experiments were performed, using four technical replicates per experiment. 120 h after adding the drugs, ATP levels were assessed with CellTiter-Glo 3D reagent (Promega) according to the manufacturer’s instructions on a BMG Labtech FLUOstar Omega microplate reader. Results were normalized to DMSO vehicle (100%). Survival data was analyzed and visualized in GraphPad Prism (version 9.3.1). IC50 values were calculated in RStudio, using the drc package (v.3.0-1).

[0169] For the Drug Screening and Combination Testing in Kidney Tumor Organoids, between 500 (for normal kidney and MRT organoids) and 2,000 (for AT / RT and SCCOHT tumoroids) small organoids were seeded per well in black 384-well plates (Corning) in 40 pL medium. Normal kidney and MRT organoids were embedded in 5% BME in kidney organoid medium (KOM). AT / RT and SCCOHT tumoroids were seeded in suspension in their specific media. For uridine rescue, either 3 pM or 30 pM uridine was added to KOM. Seeding was done with a Multi-drop Combi Reagent 8 Dispenser. Compound treatments were applied using a Tecan D300e Digital Dispenser, normalizing all wells for DMSO content. Compounds included BAY-2402234, GTX-196, AG-636, ASLAN003 (Farudodstat), PTC299, Dipyridamole, Nitrobenzylthioinosine (NBMPR), and Draflazine, tested between 0.01 nM and 10 pM. DMSO-only treated cells were negative controls. Standard dose-response studies were performed with three biological replicates and four technical replicates per model, drug combination (matrix) screens with three biological replicates. After 120 hours of treatment, ATP levels (cell viability) were measured using CellTiter-Glo 3D (Promega) on a BMG Labtech FLUOstar Omega plate reader and normalized to DMSO controls.

[0170] Flow cytometric analysis of Annexin V / DAPI positive cells:

[0171] Forthis experiment, a slightly adjusted version ofthe protocol from Calandrini et al., 2021 was used. Normal kidney (lines 16H, 57H, 60H, 71 H) and MRT organoids (lines 60T, 78T, 103T) were passaged 1 :2 and 1 :4, respectively, and plated in 75% BME droplets topped with KOM. After three (MRT organoids) to six days (normal kidney organoids), cells were re-plated in 5% BME slurry with KOM and treated with either DMSO, 400 nM MTX (Merck, M1000000) or 50 nM BAY-2402234 (DC Chemicals, DC23745). After 120 h, organoids and supernatant were harvested and made into a single-cell suspension using TrypLE Express (ThermoFisher) with Rho-kinase inhibitor Y-27632 (AbMole). Staining of single cells was done with APC-Annexin V (BD Biosciences, #550475) and DAPI (ThermoFisher, #D1306) in Annexin V binding buffer supplemented with 2.5mM Ca2+. For flow cytometric analysis of the cells, the Beckman Cytoflex LX was used. Data were subsequently analyzed with FlowJo Software (BD Biosciences, version 10). Apoptotic indices were calculated by normalizing the percentages to DMSO controls (set to 1). Live and apoptotic fractions were calculated by dividing the number of events in that gated population through the sum of all gated events (Live + Early Apoptotic + Late Apoptotic = 1).

[0172] Folinic acid / nucleoside rescue after drug treatment:

[0173] Organoids were dissociated and made into a single-cell solution using mechanical disruption. Single cells were plated at a density of 2.000-2.500 cells / pL in 5 pL 75% BME droplets topped with KOM in a flat-bottom 96 wells plate (Greiner, 655-160). For each condition, cells were plated in quadruplicate. After 48 h (78T and 103T) to 72 h (60T), treatment with DMSO, 400 nM MTX (Merck, M1000000) or BAY-2402234 (DC Chemicals, DC23745) alone or in combination with either 1x EmbryoMax Nucleosides (Sigma-Aldrich, ES-008-D) or 10 pM Folinic acid (Leucovorin, Sigma- Aldrich, #47612) was started. In all the wells, medium was refreshed daily, due to the instability of the nucleosides in the medium. All treatments lasted for 120 h. At the day of the readout, all medium was removed and 45 pL of adDF+++ together with 50 pL of the CellTiter-Glo 3D solution (Promega) was added to each well. After 5min of shaking and 30 min at RT in the dark, 80 pL of each well was transferred into a black clear bottom 396-wells plate (Corning). Luminescence was measured with the FLUOstar Omega microplate reader (BMG Labtech) and data was analyzed with GraphPad Prism software (version 9.3.1). Results were normalized to DMSO-only treated cells (100%) for each separate organoid line.

[0174] In vivo studies

[0175] For measuring the in vivo efficacy in MRT xenografts, 250.000 small size MRT organoids (78T and 103T) were harvested and implanted subcutaneously in the right flank of NOD-Scid IL2Rgnull mice, using a 1 :1 BME:Cell suspension. When tumor volumes reached 100-150 mm3, mice were put on a folic acid depleted diet (SAFE, #U8958, Version 194). After one week on the diet, mice were randomly assigned to either the MTX or saline treatment groups. Depending on the treatment group, mice received daily intraperitoneal injections of 0.75 mg / kg MTX (Emthexate PF, TEVA Pharmachemie) or saline for a total of three (78T) to four (103T) weeks.

[0176] Tumor volumes were monitored two (78T) or three (103T) times a week via caliper measurements. Mice were sacrificed when reaching humane endpoint (losing >20% body weight or having a tumor >1500 mm3). When mice reached a tumor volume exceeding 1500 mm3before the end of the treatment, this volume (>1500 mm3) was reported and maintained for that mouse until the treatment period was completed. Tumors and organs were collected for further histological analyses.

[0177] Table 2. Key resource table.

[0178]

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[0240] Example 2 - Metabolic profiling of patient-derived organoids reveals nucleotide synthesis as a metabolic vulnerability in malignant rhabdoid tumors (MRT)

[0241] Increased nucleotide biosynthesis is a hallmark and vulnerability of MRT:

[0242] Principal component analysis and hierarchical clustering demonstrated that tumoroids retain the metabolic signatures of their tumor / tissue of origin. Gene ontology enrichment analysis revealed significant upregulation of genes involved in purine and pyrimidine nucleotide biosynthesis in MRT tumoroids. LC-MS quantitative profiling showed that MRT tumoroids exhibited elevated levels of nucleotides and pathway precursors or intermediates. These results established increased nucleotide biosynthesis as a hallmark and vulnerability of MRT (data not shown).

[0243] Nucleotide Synthesis Inhibitors Selectively Impair Viability of MRT and AT / RT Tumoroids:

[0244] Drug screens were performed on three MRT, six AT / RT, four normal kidney, Wilms tumor, and additional rhabdomyosarcoma (RMS) organoid lines. Tumoroids were exposed to methotrexate (MTX; 0.1 nM-10 pM) or BAY-2402234 (BAY; 0.01 nM-1 pM) for 120 hours. After treatment, cell viability was measured by ATP detection as described in Example 1 . Both MTX and BAY-2402234 elicited dose-dependent and marked reductions in viability of MRT and AT / RT tumoroids, with IC50 values in the low nanomolar range, compared to negligible effects on normal kidney (60H, 78H, 103H) and Wilms tumor organoids (Fig. 3A, 3C). RMS organoid lines were responsive to BAY (Fig. 3D) but not to MTX (Fig. 3B). Apoptosis analysis by flow cytometric quantification of Annexin V and DAPI revealed significant induction of early and / or late apoptotic cell fractions in MRT tumoroids treated with MTX or BAY, with no harmful effect observed in normal kidney control organoids (Fig. 1 B).

[0245] Stable Isotope Tracing Demonstrates Activation of De Novo Nucleotide Synthesis: [U-13C6]-glucose isotope tracing was conducted on MRT and normal kidney organoids. After labeling, LC-MS-measured isotopologue fractions of metabolites were analyzed as described in Example 1. MRT tumoroids exhibited robust [U-13C6]-glucose incorporation into purine (ADP) and pyrimidine (UDP) nucleotides, primarily through de novo synthesis, shown by the formation of labeled isotopologues ([M+6-8]) absent in normal kidney organoids (Fig. 1 C). Treatment with MTX and BAY-2402234 (Fig. 1 D) abolished glucose incorporation into “de novo” synthesized nucleotide pools in MRT tumoroids, supporting their mechanisms as inhibitors of nucleotide synthesis.

[0246] Rescue Experiments Confirm On-Target Effects of Nucleotide Synthesis Inhibitors:

[0247] Following treatment with MTX or BAY, MRT and normal kidney organoids were supplemented with folinic acid (FA) or nucleosides, and after 120 hours the viability was assayed as described in Example 1 . Folinic acid and nucleoside supplementation fully rescued MTX-induced cell death in MRT tumoroids, while nucleosides alone rescued BAY-induced cell death, confirming the cytotoxicity arises directly through inhibition of nucleotide biosynthesis, and supporting that both inhibitors work through the proposed mechanism of action.

[0248] Drug Screening and Combination Testing in Kidney Tumor Organoids:

[0249] Following treatment with a panel of metabolic inhibitors as described in Example 1 — including BAY- 2402234, GTX-196, AG-636 (Fig. 2A), ASLAN003 (Fig. 2B), PTC299 (Fig. 2C)— MRT and AT / RT tumoroids exhibited strong, dose-dependent reductions in cell viability, with IC50 values for BAY- 2402234 and related DHODH inhibitors in the low nanomolar range. In contrast, normal kidney organoids were minimally affected, even at the highest tested concentrations (Fig. 2A-C). SCCOHT tumoroids also demonstrated sensitivity to select DHODH inhibitors (Figures 5D, 6G, 6H). Importantly, addition of uridine (3 pM or 30 pM) partially to fully rescued the cytotoxic effects of BAY- 2402234 in MRT organoids, confirming that cell death resulted from specific inhibition of de novo pyrimidine nucleotide synthesis. Drug combination (matrix) screening with DHODH inhibitors (GTX- 196, BAY-2402234) and hENT inhibitors (Dipyridamole, NBMPR, Draflazine) further identified synergistic interactions between DHODH inhibitors and hENT inhibitors in MRT models in the presence of uridine. These results demonstrated the selectivity and potential therapeutic utility of targeting the nucleotide synthesis pathway in rhabdoid and related tumor organoids. Methotrexate and BAY-2402234 Screening in Organoid Models:

[0250] IC50 values for both compounds were substantially higher in RMS organoids, with incomplete inhibition of cell viability at concentrations effective in MRT models. An overview of the exact IC50 values of MTX and BAY for each tumoroid and organoid model shown in Fig. 3A-D are shown in Table 3. These results were consistent across multiple RMS and Wilm’s tumors replicates and confirmed that the heightened sensitivity to nucleotide synthesis inhibition is specific to rhabdoid tumor lineages rather than a general feature of pediatric solid tumor organoids. Table 3. Treatment with MTX and BAY shows cytotoxic effects in rhabdoid tumors. An overview of the exact IC50 values of MTX and BAY for each tumoroid and organoid model shown in Fig. 3A-D. IC50 values are given in pM.

[0251] In vivo efficacy in MRT Xenografts: NOD-Scid IL2Rgnull mice engrafted with MRT tumoroids were subjected to MTX and BAY-2402234 treatment. MTX and BAY-2402234 treatment resulted in significant delays in tumor growth and reductions in proliferation (Ki67 index) in two independent MRT xenograft models relative to saline controls (Fig. 4). Body weight measurements indicated some MTX-associated toxicity, consistent with known clinical findings upon prolonged or high MTX dosing (data not shown).

[0252] Example 3 - Inhibition of de novo pyrimidine synthesis marks a vulnerability of SWI / SNF-mutant tumors in general

[0253] Using patient-derived tumoroid models, we evaluated the sensitivity of MRT to a panel of DHODHi and extended our investigation to AT / RT and SCCOHT to assess whether dependence on de novo nucleotide biosynthesis constitutes a shared metabolic vulnerability across SWI / SNF-deficient pediatric cancers. To better capture the in vivo metabolic context, we profiled plasma and tumor interstitial fluid (TIF) from orthotopically implanted MRT-bearing mice. This approach revealed TME- derived nutrients that drive resistance to DHODHi and enabled the identification of rational combination strategies with hENT inhibitors to overcome this resistance - insights not achievable using standard in vitro culture conditions.

[0254] Analysis of the average expression of genes involved in de novo nucleotide biosynthesis across subtypes of various pediatric tumor types revealed strong tumor-type-specific clustering, with SCCOHT, MRT, AT / RT-MYC, and AT / RT-TYR exhibiting the highest expression levels of nucleotide synthesis genes (Fig. 5A). Rhabdomyosarcoma (RMS) samples displayed intermediate expression, while the average expression in Wilms tumors, Medulloblastoma (MB) and pediatric high-grade glioma (pHGG) samples was lowest. Exceptions were the blastemal subtype of Wilms tumor and the sonic hedgehog (SHH) subtype of MB that clustered more closely with the highly expressing SWI / SNF-deficient tumors, rather than with other Wilms tumors or MB (Fig. 5A).

[0255] Given that elevated expression of de novo nucleotide biosynthesis genes emerged as a common feature among SWI / SNF-mutant tumors, we next assessed the efficacy of the most potent DHODHi in patient-derived AT / RT and SCCOHT tumoroids. Consistent with findings in MRT, both AT / RT and SCCOHT tumoroids displayed marked sensitivity to treatment with GTX-196 and BAY, with IC50 values in the low nanomolar range (Figure 6G-H). Importantly, the marked sensitivity of SWI / SNF-mutated tumoroids to DHODHi treatment was associated with a significant increase in apoptotic cell death relative to normal kidney controls (Figure 5D). Together, these results support the notion that SWI / SNF-deficient tumors share a metabolic dependency on de novo nucleotide synthesis, and that targeting of DHODH may represent a tissue-agnostic therapeutic vulnerability in this subclass of pediatric cancers.

[0256] Description of the drawings

[0257] Fig. 1A - The de novo nucleotide synthesis is a metabolic vulnerability of rhabdoid tumors with a SMARCB1 mutation that can be targeted with DHODHi BAY-2402234. Dose-response curves of BAY-2402234 for the indicated organoid and tumoroid cultures. Data points are represented as the mean ± SD of three independent experiments, each consisting of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dashed horizontal line represents a viability of 50% (IC50).

[0258] Fig. 1 B - The de novo nucleotide synthesis is a metabolic vulnerability of rhabdoid tumors with a SMARCB1 mutation that can be targeted with DHODHi BAY-2402234. Bar graphs representing live, early apoptotic, and late apoptotic cell fractions of SMARCB1 -mutated MRT and normal kidney organoids upon treatment with 50 nM BAY for 120 h. The means ± SEM of n = 3 kidney organoid models are plotted. P values were generated by performing multiple paired Student’s t test.

[0259] Fig. 1 C - The de novo nucleotide synthesis is a metabolic vulnerability of rhabdoid tumors with a SMARCB1 mutation that can be targeted with DHODHi BAY-2402234. Incorporation of glucosederived 13C in UDP overtime in three normal kidney organoids and three SMARCB1 -mutated MRT tumoroids after 24 hours of culturing in [U-13C6]-glucose. The effect of organoid type (normal kidney or SMARCB1 -mutated MRT) on glucose incorporation over time was analyzed using a linear model with an interaction term of class and time. Significant interaction between class and time was defined as an improved model fit with interaction term of class and time over a model lacking this interaction. Student’s t tests were performed on individual timepoints.

[0260] Fig. 1 D - The de novo nucleotide synthesis is a metabolic vulnerability of rhabdoid tumors with a SMARCB1 mutation that can be targeted with DHODHi BAY-2402234. Isotope distribution of UDP in three SMARCB1 -mutated MRT tumoroids after 24 hours of culturing in [U-13C6]-glucose in the presence and absence of 5 nM BAY. Student’s t test between Normal and SMARCB1 -mutated MRT was performed on mean peak area of the sum of all isotopologues of individual cell lines. (*, p < 0.05; **, p < 0.01 ; ***, p< 0.001 ; ****, p < 0.0001).

[0261] Fig. 2A - SMARCB1 -mutated MRT tumoroids are sensitive to several DHODH inhibitors. Doseresponse curves of the DHODH inhibitor AG-636 for the indicated normal kidney organoid and SMARCB1 -mutated MRT tumoroid cultures. Data points are represented as the mean ± SD of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dashed horizontal line represents a viability of 50% (IC50).

[0262] Fig. 2B - As in Fig. 2A, showing the dose-response curves of the DHODH inhibitor Farudodstat Fig. 2C - As in Fig. 2A, showing the dose-response curves of the DHODH inhibitor PTC299.

[0263] Fig. 3A - Treatment with MTX and BAY shows cytotoxic effects in rhabdoid tumors. Dose response curves of MTX for the indicated 3D tumoroid cultures. Clinical characteristics of our patient-derived models can be found in Table 1 . Data points of Wilms tumoroids are represented as the mean ± SD of three independent experiments, each consisting of quadruplicate measurements. Data points of normal kidney organoids and SMARCB1 -mutated MRT tumoroids are represented as the mean ± SD of n = 3 different models for which three independent experiments were performed, each consisting of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dashed horizontal line represents a viability of 50% (IC50).

[0264] Fig. 3B - As in Fig. 3A, showing datapoints for RMS tumoroids. Fig. 3C - Treatment with MTX and BAY shows cytotoxic effects in rhabdoid tumors. Dose response curves of BAY for the indicated 3D tumoroid cultures. Clinical characteristics of our patient-derived models can be found in Table 1 . Data points of Wilms tumoroids are represented as the mean ± SD of three independent experiments, each consisting of quadruplicate measurements. Data points of normal kidney organoids and SMARCB1 -mutated MRT tumoroids are represented as the mean ± SD of n = 3 different models for which three independent experiments were performed, each consisting of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dashed horizontal line represents a viability of 50% (IC50).

[0265] Fig. 3D - As in Fig. 3C, showing datapoints for RMS tumoroids.

[0266] Fig. 4A - Experimental overview of in vivo BAY-2402234 testing. Mice were subcutaneously injected with SMARCB1 -mutated MRT organoids. When tumor volumes reached 200 mm3, mice received oral administration of 4 mg / kg BAY or saline vehicle for a duration of four weeks.

[0267] Fig. 4B - BAY-2402234 delays SMARCB1 -mutated MRT growth in vivo. Tumor growth of SMARCB1 -mutated MRT model 103T PDX mice (n = 6 mice per treatment arm) treated with either saline vehicle or 4 mg / kg BAY. Data are represented as means ± SEM. P value was calculated using a two-tailed unpaired Student’s t test.

[0268] Fig. 4C - BAY-2402234 delays SMARCB1 -mutated MRT growth in vivo. Tumor growth of SMARCB1 -mutated MRT model 78T2 PDX mice (n = 3 for vehicle, n = 4 for BAY) treated with either saline vehicle or 4 mg / kg BAY. Data are represented as means ± SEM. P value was calculated using a two-tailed unpaired Student’s t test.

[0269] Fig. 5A - Inhibition of de novo pyrimidine synthesis enzyme DHODH marks a vulnerability of SWI / SNF-mutant tumors. Z score-based heatmap visualizing the average expression of genes involved in de novo nucleotide biosynthesis for each tumor subtype. Annotation colors denote tumor entity, subtype and SWI / SNF-mutation status. The number of samples per tumor subtype is indicated beneath the heatmap.

[0270] Fig. 5B - Dose-response curves of DHODH inhibitors AG-636, Farudodstat (ASLAN003), PTC299, GTX-196 and BAY-2402234 for patient-derived MRT tumoroid cultures. Data points are represented as the mean ± SD of three independent MRT models, with each model measured in quadruplicate. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC50).

[0271] Fig. 5C - Bar graphs representing live, early apoptotic, and late apoptotic cell fractions of MRT tumoroids and normal kidney organoids upon treatment with 50 nM GTX-196 or DMSO vehicle control for 120 h. The means ± SD of n = 3 models are plotted. P values were generated by performing multiple paired Student’s t tests (***, p < 0.001 ; ***, p < 0.0001).

[0272] Fig. 5D - Representative flow cytometry histograms depicting Annexin V surface expression as a marker of apoptosis in normal kidney organoids and AT / RT and SCCOHT tumoroid cultures following treatment with 50 nM GTX-196 or DMSO vehicle control.

[0273] Fig. 6A - Inhibition of de novo pyrimidine synthesis enzyme DHODH marks a vulnerability of SWI / SNF-mutant tumors (extended). Dose-response curves of DHODH inhibitors BAY-2402234 (panel A), PTC299 (panel B), Farudodstat (ASLAN003) (panel C); GTX-196 (panel D); and AG-636 (panel E) for the indicated normal kidney organoid and MRT tumoroid cultures. Data ponts are represented as the mean ± SD of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC50).

[0274] Fig. 6B - As in Fig. 6A, for the DHODH inhibitor PTC299.

[0275] Fig. 6C - As in Fig. 6A, for the DHODH inhibitor Farudodstat (ASLAN003).

[0276] Fig. 6D - As in Fig. 6A, for the DHODH inhibitor GTX-196.

[0277] Fig. 6E - As in Fig. 6A, for the DHODH inhibitor AG-636.

[0278] Fig. 6F - Scatterplots showing the individual IC50 of each MRT tumoroid model, grouped per DHODH inhibitor. Mean ± SD are also plotted.

[0279] Fig. 6G - Dose-response curves of BAY-2402234 for the indicated normal kidney organoid and AT / RT and SCCOHT tumoroid cultures. Data ponts are represented as the mean ± SD of three independent experiments, each consisting of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC50).

[0280] Fig. 6H - Dose-response curves of GTX-196 for the indicated normal kidney organoid and AT / RT and SCCOHT tumoroid cultures. Data points are represented as the mean ± SD of three independent experiments, each consisting of quadruplicate measurements. Data are normalized to DMSO vehicle (100%). The grey dotted horizontal line represents a viability of 50% (IC50).

Claims

Claims1 . An inhibitor of the human dihydroorotate dehydrogenase (DHODH) enzyme for use in treating a cancer in a patient, wherein the cancer is characterized by a low or absent functional activity of a subunit of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex.

2. The inhibitor for use according to claim 1 , wherein the subunit of the SWI / SNF complex is a SMARC subunit.

3. The inhibitor for use according to claim 2, wherein the SMARC subunit is SMARCB1 , SMARCA4, SMARCC1 , SMARCC2, SMARCD1 , or SMARCA2, preferably SMARCB1 or SMARCA4, more preferably SMARCB1 .

4. The inhibitor for use according to any one of claims 1-3, wherein the functional activity is absent.

5. The inhibitor for use according to any one of claims 1-4, wherein the patient has, or is suspected of having, a cancer selected from the group consisting of malignant rhabdoid tumors (MRT), atypical teratoid / rhabdoid tumors (AT / RT), epithelioid sarcomas, synovial sarcomas, undifferentiated sarcomas with or without rhabdoid features, ovarian hypercalcaemic small cell carcinomas, non-small cell lung cancer, endometrial undifferentiated carcinoma and sarcoma, gastrointestinal undifferentiated carcinomas, clear cell renal cell carcinoma, prostate cancer, extra skeletal myxoid chondrosarcomas, renal medullary carcinomas, mucinous carcinomas of the pancreas, malignant peripheral nerve sheath tumors, schwannomas, familial and sporadic schwannomatosis, cribriform neuroepithelial tumors, embryonal central nervous system tumors with or without rhabdoid features, choroid plexus carcinomas, teratoma, primitive neuroectodermal tumors, poorly differentiated chordomas, non-Hodgkin lymphoma, and chronic myeloid leukemia.

6. The inhibitor for use according to claim 5, wherein the patient has, or is suspected of having, malignant rhabdoid tumors (MRT) and / or atypical teratoid / rhabdoid tumors (AT / RT).

7. The inhibitor for use according to claim 6, wherein the patient is assessed prior to commencement of treatment with the inhibitor, wherein the assessment of the patient comprises assessing in a sample of cells obtained from the patient the functional activity of a subunit of the SWI / SNF complex, preferably of SMARCB1 or SMARCA4, and wherein low or absent functional activity indicates that treatment with the inhibitor can be commenced.

8. The inhibitor for use according to claim 7, wherein the sample of cells are cancer cells.

9. The inhibitor for use according to claim 7 or 8, wherein assessing the functional activity of the subunit of the SWI / SNF complex comprises at least one of: i) measuring the amount of SMARCB1 or SMARCA4 protein, ii) measuring the amount of SMARCB1 or SMARCA4 mRNA, and iii) determining the sequence encoding SMARCB1 or SMARCA4, preferably determining the genomic DNA sequence, cDNA sequence or mRNA sequence encoding SMARCB1 or SMARCA4.

10. The inhibitor for use according to any one of claims 7-9, wherein assessment of the patient comprises diagnosing the type of cancer from which the patient is suffering.11 . The inhibitor for use according to any one of claims 1-10, wherein the DHODH inhibitor is selected from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, GTX 196, ASLAN003, JNJ- 74856665, and PTC299, preferably from the group consisting of BAY-2402234, AG636 / AUR-108, Farudodstat, ASLAN003, and PTC299, more preferably PTC299 or BAY-2402234, more preferably BAY-2402234.

12. The inhibitor for use according to any one of claims 1-11 , wherein the inhibitor is administered by parenteral, intratumoral, oral, intravenous, transdermal or intramuscular administration, preferably intrathecal or intraventricular administration.

13. The inhibitor for use according to any one of claims 1-12, wherein the DHODH inhibitor is administered at a dose of between 4-500 mg / kg per dose.

14. A pharmaceutical formulation comprising the inhibitor for use according to any one of claims 1-13, wherein the inhibitor in the formulation has a concentration of between 1 pM and 1 mM.

15. A method of treating cancer in a subject, wherein the cancer is characterized by a low or absent functional activity of a subunit of the SWItch / Sucrose Non-Fermentable (SWI / SNF) chromatin-remodeling complex, comprising administering to the subject in need thereof the inhibitor as defined in any one of claims 1-13, or the pharmaceutical formulation as defined in claim 14.

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